This invention relates to methods and strategies for determining the enantiomeric purity of chiral compounds through the use of the optical activity of enantiomers, spectroscopy, polarimetry, and chemometric modeling.
Chiral molecules are molecules that are isomers that are mirror images and yet cannot be superimposed on each other. They are enantiomers. The enantiomers differ from achiral compounds in that enantiomers have no plane of symmetry. The general rule is any structure that doesn't have a plane of symmetry is chiral, and any structure that has a plane of symmetry cannot exist as two enantiomers.
On the molecular level, if a molecule contains a carbon atom that is bound to four different groups or atoms it will not have a plane of symmetry and must be chiral. Such a carbon atom is said to be a stereogenic or chiral center. Other formations such as a helix can be chiral, also. A chiral compound in either form can be separated or synthesized to its pure state. A mixture of 50% of each of the enantiomers is a racemate.
Another property of enantiomers or chiral compounds is the fact that they are capable of rotating the plane of oscillation of a linearly polarized light beam upon passage through a medium in either crystalline, liquid, or solution form. The amount of rotation is proportional to the percent composition of each enantiomer. Therefore a racemate mixture or 50%-50% mixture is optically inactive because each type of enantiomer cancels out the other's effect on the rotation of the polarized light. In contrast, one form will rotate the light the maximum possible in one direction while the other enantiomerically pure form will rotate the light in the opposite direction the same maximum value.
Enantiomers can be classified as R and S, but can also be identified by the direction of which way they rotate light. If the compound rotates the light to the right, the convention says that the enantiomer is (+) or dextrotary. If the compound rotates the light to the left, then this enantiomer is designated as (−) or laevorotatory.
Many biological systems are configured chirally. The difference between two enantiomers can have enormous consequences because many important biological molecules are chiral. Amino acids are the building blocks of proteins and enzymes in all terrestrial life and have the formula +H3NCH(R)CO2−. Note that the C atom is bonded to four different groups, making it a chiral center. All naturally occurring amino acids exist as only one of the two possible enantiomers, meaning that all proteins and enzymes are also chiral. Sugars, polysaccharides and peptides are also chiral.
Drug activity results from pharmacological and pharmokinetic processes by which it enters, interacts with, and leaves the body. Many examples exist in which enantiomers show marked differences in their bioavailability, distribution, metabolic, and permeability behavior in which stereochemical parameters determine the fundamental action of the compound on the biochemical systems of a living organism. For instance, the β-blocker propranol or the cardotonic agent verapamil have different efficacy when administered as racemates and not as the active enantiomer. The R form of PROZAC is the active configuration of the drug while the S form is inactive. In the late fifties and early sixties, the racemate of the drug Thalidomide was given to pregnant women to control nausea and as a sedative. The R configuration was responsible for desired effect; however, unknown to the medical community, the S form was later found to be a teratogen. A teratogen is a chemical that causes developmental defects in unborn infants. This drug was responsible for hundreds of thousands of birth defects before this fact was discovered.
Economic and safety interests are obvious and are paramount in the development of new chiral substances and technological advances. Single-enantiomer sales have shown a steady annual increase. Consequently, many of the top selling drugs are marketed as single enantiomer compounds.
The need for improved strategies for the assessment of enantiomeric purity arises from increased pressure on the pharmaceutical industry by government agencies for documentation on the pharmacological effects of individual enantiomers and the simultaneous demand in drug development for determination of enantiomeric excess in large combinatorial libraries. The significance of chirality on almost any pharmacological process is well documented. Also, economically, chiral technologies in other fields such as agrochemicals, food additives, fragrances, new materials and catalysts have come to the forefront. In conjunction with an ever increasing demand for enantiomerically pure compounds efficient strategies for analysis and preparation of chiral molecules are required. For high throughput screening strategies, slow chromatographic methods are not attractive. Rapid spectroscopic techniques are the most desirable.
Several methods are presently used to determine enantiomeric purity. The traditional and most commonly known is polarimetry. As mentioned earlier, the plane of oscillation of linearly polarized light is rotated in proportion to the enantiomeric make-up of a mixture of R and S enantiomers. Optical activity is observed and measured by means of an instrument known as a polarimeter, which was first used as a chemical instrument in about 1816. Light from a monochromic light source is passed through a polarizer, which linearly polarizes the light. It is then allowed to pass through a sample tube which contains the sample to be analyzed and then through a second polarizer. The angle of rotation is determined by maximizing the light passing through the second polarizer. This position would coincide with the plane of oscillation of the polarized light which would correlate with the angle of rotation. The polarimeter can be used to determine if a given substance is chiral or achiral by observing if it rotates light. The enantiomeric composition of chiral samples can be ascertained either quantitatively or qualitatively, since the rotation is correlated with the enantiomeric purity or composition. It can also be used to investigate equilibrium systems and reaction mechanisms. It is popular since it is a reliable, relatively simple, and well established technique. The disadvantage of polarimeter is that it is not very sensitive and is affected by chiral impurities.
Another strategy is the covalent synthesis and detection of diastereomers using enantiomerically pure derivatizing agents. In this scenario, the enantiomers are derivatized and then, since diastereomers have different chemical and physical properties, the enantiomeric purity can be detected using several techniques which include nuclear magnetic resonance spectroscopy.
Other classical methods of enantiomeric purity include the detection of transient diastereomeric interactions by NMR using chiral shift reagents, and the use of chiral stationary phases in chromatography. The last technology at present is the most desirable technique in terms of speed, accuracy, and adaptability of the ones mentioned.
Multivariate regression modeling (“MRM”) is widely used in chemistry as a means of correlating spectral data with known compositional changes.